This invention relates to mammalian cDNAs which encode SPARC-related proteins and to the use of the cDNAs and the encoded proteins in the diagnosis and treatment of cell proliferative disorders.
Phylogenetic relationships among organisms have been demonstrated many times, and studies from a diversity of prokaryotic and eukaryotic organisms suggest a more or less gradual evolution of molecules, biochemical and physiological mechanisms, and metabolic pathways. Despite different evolutionary pressures, the proteins of nematode, fly, rat, and man have common chemical and structural features and generally perform the same cellular function. Comparisons of the nucleic acid and protein sequences from organisms where structure and/or function are known accelerate the investigation of human sequences and allow the development of model systems for testing diagnostic and therapeutic agents for human conditions, diseases, and disorders.
The interaction of a cell with its surrounding extracellular matrix (ECM) influences cell behavior. The ECM, composed of fibrous proteins, proteoglycans and glycoproteins, fills the extracellular space with an elaborate protein network that establishes cellular shape, adhesion, detachment, motility, growth, division, and differentiation. Variations in the composition of the ECM determine the distinctive character of tissues and account for differences in strength and flexibility of connective tissues such as skin, bone, tendon, ligament and cartilage. Restructuring of the ECM accompanies embryonic development, tissue remodeling, angiogenesis, and wound healing.
Glycoproteins of the ECM typically contain multiple domains that mediate protein-protein interactions among ECM proteins and between ECM proteins and cell surface receptors. They frequently contain a variety of post-translational modifications that are required for their function, including covalently attached N- and O-linked complex-carbohydrates, phosphorylated serine and threonine residues and sulfated tyrosine residues. SPARC, an abbreviation for secreted protein acidic and rich in cysteine, also termed osteonectin, BM-40, and 43K protein, is an ECM glycoprotein that carries out multiple functions (Lane and Sage (1994) FASEB J 163-173; Motamed (1999) Int J Biochem Cell Biol 31:1363-1366). It has a molecular weight of 33 kDa in the absence of post-translational modifications, is 303 amino acids in length, and contains covalently attached N-linked complex-type carbohydrate and a signal peptide of 17 amino acids. Among its roles, SPARC modulates cell shape, adhesion, and migration of cells. Cells which over-express SPARC have a rounded morphology, whereas cells which under-express SPARC flatten. Acting as an anti-adhesin, SPARC disrupts interactions of cells with other ECM proteins. It is expressed during embryogenesis, tissue remodeling and repair. SPARC is present at high levels in developing bone and teeth, where it may be involved in calcification and calcium ion binding and may function in the development of ossified and mineralized tissues. SPARC is also present at high concentrations in activated platelets and megakaryocytes. SPARC binds cytokines, divalent cations, several collagen types, hydroxyapatite, albumin, thrombospondin and cell membranes on platelets and endothelial cells. It modulates the responses of cells to cytokines and inhibits the progression of the cell cycle from G1 to S phase.
SPARC is made up of three domains, which individually have been shown to carry out specific functions (Motamed, supra). The acidic domain binds Ca2+, inhibits cell spreading and chemotactic responses to growth factors, modulates levels of plasminogen activator inhibitor-1, fibronectin, and thrombospondin-1. The cysteine-rich domain has homology with follistatin, an inhibitor of transforming growth factor b-like cytokines, and also shows similarity to serpin-type protease inhibitors and epidermal growth factor (EGF)-like motifs. This domain controls cell proliferation, angiogenesis, and disassembly of focal adhesions that link the ECM to the actin cytoskeleton. The extracellular calcium-binding domain contains an EF-hand motif, binds to cells and several types of collagen, induces matrix metalloproteinases, inhibits cell spreading and proliferation, and controls focal adhesions. Binding of collagen is dependent on Ca2+ and the state of protein glycosylation.
During normal development, angiogenesis, and wound healing, SPARC modulates the effects of a variety of growth factors involved in cell cycle control, cell migration, and proliferation. Perturbed cellular regulation by growth factors is associated with altered levels of SPARC expression and pathological processes in various tissues. For example, SPARC shows high levels of expression in lesions of atherosclerosis compared to normal vessels (Raines et al. (1992) Proc Natl Acad Sci 89:1281-1285). It controls the activity of platelet-derived growth factor (PDGF), which promotes cell migration, proliferation, and cellular metabolic changes. SPARC binds to PDGF and inhibits its interaction with receptors. By regulating the availability of PDGF in response to vascular injury, SPARC may control proliferative repair processes. SPARC delays the entry of aortic endothelial cells into S phase and may facilitate withdrawal from the cell cycle in response to injury or developmental signals (Funk and Sage (1991) Proc Natl Acad Sci 88:2648-2652). SPARC may also play a role in the calcification of atherosclerotic plaques (Watson et al. (1994) J Clin Invest 93:2106-2113).
SPARC shows high levels of expression in brain tumor cells in gliomas where it controls the activity of vascular endothelial growth factor (VEGF), the principal angiogenic growth factor identified in human astroglial tumors (Vajkoczy et al. (2000) Int J Cancer 87:261-268). VEGF participates in a signal-transduction pathway that mediates glioma angiogenesis through stimulation of tyrosine phosphorylation and activation of mitogen-activated protein kinases. SPARC binds to VEGF and inhibits its association with cell-surface receptors. In addition, the anti-adhesive properties of SPARC and its ability to induce and activate proteolytic enzymes that degrade the ECM may also play roles in promoting cell migration and tumor cell infiltration into surrounding tissue.
Overexpression of SPARC is also associated with osteoarthritis and rheumatoid arthritis (Nakamura et al. (1996) Arthritis and Rheumatism 39:539-551). High levels of SPARC are found in cartilage and synovial fluids of patients with osteoarthritis or rheumatoid arthritis compared to levels in normal cartilage. Levels of SPARC increase in articular chondrocyte cultures in response to transforming growth factor b1 and bone morphogenetic protein 2 and decrease in response to inflammatory cytokines, IL-1b, IL-1a, tumor necrosis factor a, lipospolysaccharide, phorbol myristate acetate, basic fibroblast growth factor, and dexamethasone. SPARC activates expression of matrix metalloproteinases in synovial fibroblasts and may play roles in the destruction and repair of cartilage.
In addition, aberrant expression of SPARC is associated with a number of other diseases. SPARC shows high levels of expression in breast, ovarian and prostate cancer where it may facilitate tumor progression through control of cell adhesion, growth factors and matrix metalloproteinase activity (Gilles et al. (1998) Cancer Res 58:5529-5536; Porter et al. (1995) J Histochem Cytochem 43:791-800; Brown et al. (1999) Gynecol Oncol 75:25-33; Thomas et al. (2000) Clin Cancer Res 6:1140-1149). Elevated expression of SPARC is associated with Scleroderma (Unemori and Amento (1991) Curr Opin Rheumatol 3:953-959), human lens cataracts (Kantorow et al. (2000) Mol Vis 6:24-29) and ECM deposits in renal disease (Bassuk et al. (2000) Kidney Int 57:117-128). The discovery of mammalian cDNAs encoding SPARC-related proteins satisfies a need in the art by providing compositions which are useful in the diagnosis and treatment of cell proliferative disorders.
The invention is based on the discovery of mammalian cDNAs which encodes mammalian SPARC-related proteins, SPARC-1 and SPARC-2, which are useful in the diagnosis and treatment of atherosclerosis, anaplastic oligodendroglioma, astrocytoma, oligoastrocytoma, glioblastoma, meningioma, ganglioneuroma, neuronal neoplasm, multiple sclerosis, Huntington""s disease, breast adenocarcinoma, prostate adenocarcinoma, stomach adenocarcinoma, metastasizing neuroendocrine carcinoma, nonproliferative fibrocystic and proliferative fibrocystic breast disease, gallbladder cholecystitis and cholelithiasis, osteoarthritis, and rheumatoid arthritis.
The invention provides an isolated mammalian cDNA or a fragment thereof encoding mammalian proteins or portions thereof selected from the amino acid sequences of SEQ ID NO:1 or SEQ ID NO: 2, a variant having at least 56% identity to the amino acid sequences of SEQ ID NO:1 or SEQ ID NO: 2, antigenic epitopes of SEQ ID NO:1 or SEQ ID NO:2, oligopeptides of SEQ ID NO:1 or SEQ ID NO:2, and biologically active portions of SEQ ID NO:1 or SEQ ID NO:2.
The invention also provides an isolated mammalian cDNA or the complement thereof selected from the nucleic acid sequences of SEQ ID NO:3, a variant having at least 83% identity to the nucleic acid sequence of SEQ ID NO:3, a fragment of SEQ ID NOs:4-13, and an oligonucleotide of SEQ ID NO:3. The invention additionally provides a composition, a substrate, and a probe comprising the cDNA, or the complement of the cDNA, encoding SPARC-1. The invention further provides a vector containing the cDNA, a host cell containing the vector and a method for using the cDNA to make SPARC-1. The invention still further provides a transgenic cell line or organism comprising a vector containing the cDNA encoding SPARC-1. The invention additionally provides a mammalian fragment or the complement thereof selected from the group consisting of SEQ ID NOs:14-19. In one aspect, the invention provides a substrate containing at least one of these fragments. In a second aspect, the invention provides a probe comprising the fragment which can be used in methods of detection, screening, and purification. In a further aspect, the probe is a single stranded complementary RNA or DNA molecule.
The invention also provides an isolated mammalian cDNA or the complement thereof selected from the group consisting of nucleic acid sequences of SEQ ID NO:20, a variant having 84% identity to the nucleic acid sequence of SEQ ID NO:20, a fragment of SEQ ID NOs:21-30, an oligonucleotide of SEQ ID NO:20. The invention additionally provides a composition, a substrate, and a probe comprising the cDNA, or the complement of the cDNA, encoding SPARC-2. The invention further provides a vector containing the cDNA, a host cell containing the vector and a method for using the cDNA to make SPARC-2. The invention still further provides a transgenic cell line or organism comprising a vector containing the cDNA encoding SPARC-2. The invention additionally provides a mammalian fragment or the complement thereof selected from the group consisting of SEQ ID NOs:31-40. In one aspect, the invention provides a substrate containing at least one of these fragments. In a second aspect, the invention provides a probe comprising the fragment which can be used in methods of detection, screening, and purification. In a further aspect, the probe is a single stranded complementary RNA or DNA molecule.
The invention provides a method for using a cDNA to detect the differential expression of a nucleic acid in a sample comprising hybridizing a probe to the nucleic acids, thereby forming hybridization complexes and comparing hybridization complex formation with a standard, wherein the comparison indicates the differential expression of the cDNA in the sample. In one aspect, the method of detection further comprises amplifying the nucleic acids of the sample prior to hybridization. In another aspect, the method showing differential expression of the cDNA is used to diagnose atherosclerosis or a cell proliferative disorder. In another aspect, the cDNA or a fragment or a complement thereof may comprise an element on an array.
The invention additionally provides a method for using a cDNA or a fragment or a complement thereof to screen a library or plurality of molecules or compounds to identify at least one ligand which specifically binds the cDNA, the method comprising combining the cDNA with the molecules or compounds under conditions allowing specific binding, and detecting specific binding to the cDNA, thereby identifying a ligand which specifically binds the cDNA. In one aspect, the molecules or compounds are selected from aptamers, DNA molecules, RNA molecules, peptide nucleic acids, artificial chromosome constructions, peptides, transcription factors, repressors, and regulatory molecules.
The invention provides a purified mammalian protein or a portion thereof selected from the group consisting of an amino acid sequence of SEQ ID NO:1, a variant having at least 56% identity to the amino acid sequence of SEQ ID NO:1, an antigenic epitope of SEQ ID NO:1, an oligopeptide of SEQ ID NO:1, and a biologically active portion of SEQ ID NO:1. The invention also provides a composition comprising the purified protein or a portion thereof in conjunction with a pharmaceutical carrier. The invention further provides a method of using the SPARC-1 to treat a subject with a cell proliferative disorder comprising administering to a patient in need of such treatment the composition containing the purified protein. The invention still further provides a method for using a protein to screen a library or a plurality of molecules or compounds to identify at least one ligand, the method comprising combining the protein with the molecules or compounds under conditions to allow specific binding and detecting specific binding, thereby identifying a ligand which specifically binds the protein. In one aspect, the molecules or compounds are selected from DNA molecules, RNA molecules, peptide nucleic acids, peptides, proteins, mimetics, agonists, antagonists, antibodies, immunoglobulins, inhibitors, and drugs. In another aspect, the ligand is used to treat a subject with atherosclerosis or a cell proliferative disorder.
The invention provides a purified mammalian protein or a portion thereof selected from the group consisting of an amino acid sequence of SEQ ID NO:2, a variant having at least 56% identity to the amino acid sequence of SEQ ID NO:2, an antigenic epitope of SEQ ID NO:2, an oligopeptide of SEQ ID NO:2, and a biologically active portion of SEQ ID NO:2. The invention also provides a composition comprising the purified protein or a portion thereof in conjunction with a pharmaceutical carrier. The invention further provides a method of using the SPARC-2 to treat a subject with a cell proliferative disorder comprising administering to a patient in need of such treatment the composition containing the purified protein. The invention still further provides a method for using a protein to screen a library or a plurality of molecules or compounds to identify at least one ligand, the method comprising combining the protein with the molecules or compounds under conditions to allow specific binding and detecting specific binding, thereby identifying a ligand which specifically binds the protein. In one aspect, the molecules or compounds are selected from DNA molecules, RNA molecules, peptide nucleic acids, peptides, proteins, mimetics, agonists, antagonists, antibodies, immunoglobulins, inhibitors, and drugs. In another aspect, the ligand is used to treat a subject with a cell proliferative disorder.
The invention provides a method of using a mammalian protein to screen a subject sample for antibodies which specifically bind the protein comprising isolating antibodies from the subject sample, contacting the isolated antibodies with the protein under conditions that allow specific binding, dissociating the antibody from the bound-protein, and comparing the quantity of antibody with known standards, wherein the presence or quantity of antibody is diagnostic of atherosclerosis or a cell proliferative disorder, particularly anaplastic oligodendroglioma, astrocytoma, oligoastrocytoma, glioblastoma, meningioma, ganglioneuroma, neuronal neoplasm, multiple sclerosis, Huntington""s disease, breast adenocarcinoma, prostate adenocarcinoma, stomach adenocarcinoma, metastasizing neuroendocrine carcinoma, nonproliferative fibrocystic and proliferative fibrocystic breast disease, gallbladder cholecystitis and cholelithiasis, osteoarthritis, and rheumatoid arthritis.
The invention also provides a method of using a mammalian protein to prepare and purify antibodies comprising immunizing a animal with the protein under conditions to elicit an antibody response, isolating animal antibodies, attaching the protein to a substrate, contacting the substrate with isolated antibodies under conditions to allow specific binding to the protein, and dissociating the antibodies from the protein, thereby obtaining purified antibodies.
The invention provides purified antibodies which bind specifically to polypeptides comprising the amino acid sequences selected from SEQ ID NOs:1 and 2 and fragments thereof. The invention also provides a method of using an antibody to diagnose atherosclerosis and cell proliferative disorders comprising combining the antibody comparing the quantity of bound antibody to known standards, thereby establishing the presence of atherosclerosis or a cell proliferative disorder, particularly anaplastic oligodendroglioma, astrocytoma, oligoastrocytoma, glioblastoma, meningioma, ganglioneuroma, neuronal neoplasm, multiple sclerosis, Huntington""s disease, breast adenocarcinoma, prostate adenocarcinoma, stomach adenocarcinoma, metastasizing neuroendocrine carcinoma, nonproliferative fibrocystic and proliferative fibrocystic breast disease, gallbladder cholecystitis and cholelithiasis, osteoarthritis, and rheumatoid arthritis. The invention further provides a method of using an antibody to treat atherosclerosis and cell proliferative disorders comprising administering to a patient in need of such treatment a pharmaceutical composition comprising the purified antibody.
The invention provides a method for inserting a marker gene into the genomic DNA of a mammal to disrupt the expression of the endogenous polynucleotide. The invention also provides a method for using a cDNA to produce a mammalian model system, the method comprising constructing a vector containing the cDNA selected from SEQ ID NOs:3-40, transforming the vector into an embryonic stem cell, selecting a transformed embryonic stem, microinjecting the transformed embryonic stem cell into a mammalian blastocyst, thereby forming a chimeric blastocyst, transferring the chimeric blastocyst into a pseudopregnant dam, wherein the dam gives birth to a chimeric offspring containing the cDNA in its germ line, and breeding the chimeric mammal to produce a homozygous, mammalian model system.